Nitrogen fixation in pine: Direct confirmation and location of the diazotrophic population

氮往往是自然生态系统中最具限制性的植物养分，对能够通过生物固氮提供自己供应的物种具有明显的优势。与植物相关的生物固氮可以满足某些非结瘤草本植物的部分植物氮素需求，但已知的唯一能从生物固氮中获得大部分植物氮(高达80%)的非结瘤植物包括几个甘蔗品种。甘蔗生物固氮的起因最初被认为是一种内生重氮葡萄糖乙酸杆菌菌株，但最近的研究表明，重氮营养的主要来源可能是生活在植物组织上或植物组织内的细菌群，可能包括未培养的菌株。根据甘蔗和其他草本植物中负责生物固氮的微生物是可培养的内生菌的报道，我在商业上重要的裸子植物、樟子松中寻找相似的微生物，并从表面灭菌的樟子松幼苗和树木组织的提取物中分离到几株具有显著乙炔还原活性的可培养的多粘拟青杆菌。用其中一种菌株P2b-2R处理的松树幼苗，从生物固氮中获得高达79%的叶片氮，并比对照积累了显著更多的生物量。到目前为止，我的研究结果表明：(I)如果苗木生长足够长的时间，P2b-2R的固氮作用可以获得几乎所有的叶面氮；(Ii)如果苗木生长足够长的时间，P2b-2R的生物固氮可以促进幼苗的生长；(Iii)P2b-2R的内生习性促进了生物固氮。然而，到目前为止进行的实验是探索性的，没有直接测量氮气固定，也没有排除促进幼苗生长的其他可能原因。我建议使用15N组织掺入实验来证实我们的间接证据，即P2b-2R在松树中存在BNF，并使用野生型的P2b-2R NIF-衍生物来确定这种微生物对松树幼苗生长的促进是否源于BNN。此外，由于P2b-2R在内部和外部的根、茎和针叶组织中定居，我建议确定这些与植物相关的氮固定种群中的哪一个对松树相关的生物固氮贡献最大。

Nitrogen is often the most limiting plant nutrient in natural ecosytems rendering a distinct advantage to species able to provide their own supply through biological nitrogen fixation (BNF). Plant-associated BNF can satisfy part of the plant N requirement in certain non-nodulating herbaceous species but the only known non-nodulating plants that can obtain most plant N from BNF (up to 80%) comprise a few cultivars of sugarcane.The causative agent of BNF in sugarcane was initially thought to be an endophytic strain of Gluconoacetobacter diazotrophicus, but more recent studies indicate that the primary source of diazotrophy may involve a consortium of bacteria, possibly including uncultured strains, that live on or inside plant tissues. Based on reports than the microorganism responsible for BNF in sugarcane and other herbaceous plant species were culturable endophytes, I searched for similar microorganisms in the commercially important gymnosperm, lodgepole pine and isolated several culturable Paenibacillus polymyxa strains that possessed significant acetylene reduction activity from extracts of surface-sterilized lodgepole pine seedling and tree tissues. Pine seedlngs treated with one of these strains, P2b-2R, derived up to 79% of foliar N from BNF and accumulated significantly more biomass than controls. My results so far suggest that (i) pine seedlings can derive almost all foliar N from nitrogen fixation by P2b-2R, (ii) BNF by P2b-2R causes seedling growth promotion, if seedlings are grown for a sufficiently long time and (iii) P2b-2R's endophytic habit facilitates BNF. However, the experiments conducted so far were exploratory in nature and did not measure N2 fixation directly or rule out other possible causes of seedling growth promotion. I propose to confirm our indirect evidence of BNF by P2b-2R in pine using a 15N tissue incorporation assay and to determine if pine seedling growth promotion by this microorganism results from BNF using a P2b-2R nif- derivative of the wild type. In addition, because P2b-2R colonizes internal and external root, stem and needle tissues, I propose to determine which of these plant-associated N2 fixing populations contributes most to pine-associated BNF.